WO2015027906A1 - Bifunctional fusion protein, preparation method therefor, and use thereof - Google Patents

Abstract

Provided are a bifunctional fusion protein having a CTLA4 molecule extracellular region, an Fc fragment, and a functional fragment that neutralizes TNFα activity, a gene for encoding the bifunctional fusion protein, a carrier having the gene, a host cell having the carrier, and a pharmaceutical composition having the bifunctional fusion protein. The functional fragment that neutralizes TNFα activity is a TNFR2 molecule extracellular region or an anti-TNFα single-chain antibody, wherein the CTLA4 molecule extracellular region is located at the N-terminus of the bifunctional protein, and the TNFR2 molecule extracellular region or the anti-TNFα scFv is located in the middle or the C-terminus of the bifunctional protein. The bifunctional fusion protein can simultaneously block the signal pathways of B7/CD28 and TNFα/TNFR, thereby inhibiting inflammation reactions. Comparing to single-function proteins, the present invention can lower the production cost, reduce clinically administered medicine volume and frequency, and enhance compliance of the test subjects.

Description

 Bifunctional fusion protein, preparation method and use thereof

Technical field

 The present invention relates to a bifunctional fusion protein, a preparation method thereof and use thereof, in particular, the present invention relates to a bifunctional fusion protein comprising a CTLA4 molecule extracellular region, an Fc fragment and a functional fragment having neutralizing TNFa activity, encoding the bifunctional fusion A gene of a protein, a vector containing the gene, a host cell containing the vector, and a pharmaceutical composition containing the bifunctional fusion protein. Background technique

 Rheumatoid arthritis (RA) is a chronic, progressive, systemic autoimmune disease characterized by joint synovitis with a prevalence of 0.5% to 1% in the global population. The early symptoms are swelling, pain, and difficulty in movement of the joints. As the disease progresses, the joints may be severely deformed or even disabling, and may involve other tissues and organs, causing diseases such as interstitial lung disease, pleurisy, pericarditis, and rheumatoid vasculitis. Therefore, timely and effective treatment is of great significance in preventing the progression of the disease, relieving symptoms, and improving the quality of life of patients.

Traditional RA treatments mainly use non-organic anti-inflammatory drugs, glucocorticoids and drugs that relieve the disease. With the deepening of the pathogenesis of RA, more and more biological agents have been used in the treatment of RA and have demonstrated the advantages of faster, shorter treatment cycles and fewer side effects than traditional RA treatments. The currently marketed biological agents for the treatment of RA act on a number of different targets, such as TNFa antagonists that inhibit TNFa activity, Rituximab, a monoclonal antibody against B cells, and T cell activity. Abatacept, the monoclonal antibody tocilizumab that inhibits the IL-6 signaling pathway, Anakinra, which neutralizes IL-1 activity, etc. (Vierboom M, Breedveld E, Hart BA Expert opinion on drug discovery. 2012, 7(4): 315-325). Among them, TNFa antagonists are the first-line drugs for the treatment of RA, and the market share is also the largest. TNFa is a cellular inflammatory factor that induces the secretion of other inflammatory factors by binding to its receptors TNFR1 and T FR2. Antagonists of TNFa block this binding, reduce the activity of the TNFa signaling pathway, and inhibit the inflammatory response. Currently available antagonists of TNFa are mainly soluble TNFa receptors and TNFa monoclonal antibodies. The soluble TNFa receptor fuses the extracellular domain of T FR2 to the constant region of IgG, such as Etanercept, which was marketed by Pfizer in 1998. The TNFa monoclonal antibody is a genetically engineered antibody that specifically recognizes TNFa and is capable of neutralizing T Fa. Currently, J&J's Infliximab and Golimumab are available, and UCB's Sai Tuozhu Monoclonal antibody (Certolizumab) and Abbott's Adalimumab (Lin J, Ziring D, Desai S, et al. Clinical Immunology, 2008, 126: 13-30). Clinical application shows that TNFa is antagonistic The anti-drug can effectively alleviate the inflammation of RA, relieve the radiological progress of the joint, and improve the ACR20 index of patients by 50%-70%. However, it has also been found that 20-30% of patients have unsatisfactory responses to TNFa antagonists or have a gradual decline in long-term TNFa antagonist therapy (Gibbons LJ, Hyrich KL. Biodrugs, 2009, 23 ( 2): 111-124). For this part of the patient, it is necessary to use other anti-RA drugs for treatment.

 Abnormal T cell-mediated immune response is the main pathogenesis of RA. The study found a large number of activated T cell infiltration in the synovial fluid of RA patients. These activated T cells stimulate collagen synthase cells to secrete collagenase and protease, which destroys cartilage, stimulates activation of B cells and endothelial cells, and expresses antibodies against autoantigens and inflammatory cytokines. Regulating and inhibiting the function of T cells is considered to be an effective way to treat RA (Cope AP, Arthritis research and therapy, 2008, 10(S1): 1-10). Abatacept is a biological agent for the treatment of RA by inhibiting the activation of T cells, which was developed by Bristol-Myers Squibb and launched in 2005. Abatacept fuses the extracellular domain of CTLA4 protein with the constant region of IgG, and inhibits the proliferation and activation of T cells by blocking the binding of B7 molecule to CD28. Because its mechanism is completely different from existing TNFa antagonists, Abatacept is used in the treatment of moderate to severe rheumatoid arthritis, especially in patients who have been recommended for the treatment of TNFa antagonist failure. Clinical studies have shown that after treatment with Abatacept for patients with insignificant TNFa antagonists, the ACR20, ACR50 and ACR70 indices are still 76.4%, 51.9% and 22% after 5 years, and the side effects of treatment are low ( Genovese MC, Schiff M, Luggen M, et al. The Journal of Rheumatology, 2012, 39: 1546-1554). This result indicates that Abatacept and TNFa antagonists are complementary in mechanism and effect.

 In view of the great harm of RA to patients' quality of life, it is still necessary to study a novel RA therapeutic that combines TNFa antagonists and inhibits T cell proliferation and activation. Summary of the invention

 Accordingly, the present invention provides a novel RA therapeutic agent that binds to an antagonist of TNFa and inhibits both T cell proliferation and activation.

 Accordingly, a first aspect of the invention relates to a protein, in particular to a bifunctional fusion protein comprising a CTLA4 molecule extracellular region, an Fc fragment, a functional fragment that neutralizes TNFa activity, and a linker peptide.

 In one embodiment, the extracellular region of the CTLA4 molecule is located at the N-terminus of the fusion protein.

 In one embodiment, the joining sequence of the protein is a CTLA4 molecule extracellular domain, an Fc fragment, a linker peptide, and a functional fragment that neutralizes TNFa activity.

 In one embodiment, the protein is linked in the extracellular region of the CTLA4 molecule, a linker peptide, a functional fragment that neutralizes TNFa activity, and an Fc fragment.

In one embodiment, the amino acid sequence of the extracellular region of the CTLA4 molecule is set forth in SEQ ID NO: 1. In one embodiment, the Fc fragment is selected from the group consisting of IgG, IgA, IgD, IgE, Fc fragments of IgM, and combinations and hybrids thereof. In one embodiment, the amino acid sequence of the Fc fragment is set forth in SEQ ID NO: 2.

 In one embodiment, the amino acid sequence of the linker peptide is from 1 to 50 amino acids in length, for example, the amino acid sequence of the linker peptide is 10, 15, 20, 21, 22, 23, 24, 25 or 30 amino acids in length, In one embodiment, the linker peptide has an amino acid sequence of from 1 to 25 amino acids in length, and in one embodiment, the linker peptide has an amino acid sequence length of 15 amino acids, and in one embodiment, the linker peptide The amino acid sequence is shown in SEQ ID NO: 3.

 In one embodiment, the functional fragment that neutralizes T Fa activity is the extracellular region of the T FR2 molecule. In one embodiment, the amino acid sequence of the extracellular region of the T FR2 molecule is set forth in SEQ ID NO: 4. In one embodiment, the functional fragment that neutralizes TNFa activity is a monoclonal antibody against TNFa or a functional fragment thereof, a chimeric antibody, a humanized antibody, a fully human antibody, a bispecific antibody. In one embodiment, the monoclonal antibody is an IgG, IgA, IgD, IgE, IgM antibody or a hybrid thereof. In another embodiment, the monoclonal antibody is IgG. In one embodiment, the functional fragment is a single domain antibody, a single chain antibody, a single chain variable fragment (scFv), a Fab fragment or an F(ab')2 fragment. In one embodiment, the functional fragment that neutralizes TNFa activity is a single chain antibody against TNFa. In one embodiment, the amino acid sequence of the anti-TNFa single chain antibody is set forth in SEQ ID NO: 5.

 In one embodiment, the amino acid sequence of the protein is set forth in SEQ ID NO: 6 or 7, or the sequence set forth in SEQ ID NO: 6 or 7 is replaced, deleted or added with one or more amino acid residues. An equivalent amino acid sequence, such as 2, 3, 4, 5, 10, 15, 20, 30, 50 amino acid residues, or at least 70% identical and identical to the SEQ ID NO: 6 or 7 sequence Functional amino acid sequence, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% are the same 'I'.

 In one embodiment, the amino acid sequence of the protein is set forth in SEQ ID NO: 8 or 9, or the sequence set forth in SEQ ID NO: 8 or 9 is replaced, deleted or added with one or more amino acid residues. An equivalent amino acid sequence, such as 2, 3, 4, 5, 10, 15, 20, 30, 50 amino acid residues, or at least 70% identical and identical to the SEQ ID NO: 8 or 9 sequence Functional amino acid sequence, such as at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% are the same 'I'.

A second aspect of the invention relates to a gene encoding the protein according to the first aspect. In one embodiment The nucleotide sequence of the gene is shown in SEQ ID NO: 10 or 11.

 A third aspect of the invention relates to a gene encoding the protein according to the first aspect. In one embodiment, the nucleotide sequence of the gene is as set forth in SEQ ID NO: 12 or 13.

 A fourth aspect of the invention relates to a recombinant vector operably linked to the gene according to the second aspect or the third aspect. In certain embodiments, the vector is a eukaryotic expression vector, and in certain embodiments, the vector is a vector X0GC engineered to have two expression cassettes.

 A fifth aspect of the invention relates to a host cell comprising the recombinant vector according to the fourth aspect.

 A sixth aspect of the invention relates to a method of producing a protein according to the first aspect, the method comprising:

(1) cloning the gene of the second aspect or the third aspect into a eukaryotic expression vector and transfecting it into a host cell for expression;

 (2) Purification of the protein. In one embodiment, the eukaryotic expression vector is X0GC, and in one embodiment, the host cell is HEK293-T and CHO.

 A seventh aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the protein according to the first aspect.

 An eighth aspect of the invention relates to the protein according to the first aspect or the pharmaceutical composition according to the seventh aspect, in the preparation of a medicament for preventing or treating an immune disease, a rejection reaction, and a cardiovascular disease use.

 In one embodiment, the protein is used to prepare a medicament for preventing or treating a disease: rheumatoid arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's Disease, systemic vasculitis, dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, lupus erythematosus, idiopathic thrombocytopenic purpura, glomerulonephritis, gout, organ transplantation Rejection, asthma or atherosclerosis.

 In one embodiment, the protein is used to prepare a medicament for preventing or treating a disease: osteoarthritis, psoriatic arthritis, gouty arthritis, juvenile rheumatoid arthritis, septic arthritis.

 A ninth aspect of the invention relates to the protein according to the first aspect or the pharmaceutical composition according to the seventh aspect, which is for use in the prevention or treatment of an immune disease, a rejection reaction, and a cardiovascular disease.

 In one embodiment, the protein or pharmaceutical composition for preventing or treating rheumatoid arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, system Vasculitis, dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, lupus erythematosus, idiopathic thrombocytopenic purpura, glomerulonephritis, gout, rejection of organ transplants, Asthma or atherosclerosis.

In one embodiment, the protein or pharmaceutical composition is for use in preventing or treating osteoarthritis, psoriasis Hyperinflammation, gouty arthritis, juvenile rheumatoid arthritis, septic arthritis.

 A tenth aspect of the invention relates to a method for preventing or treating an immune disease, a rejection, and a cardiovascular and cerebrovascular disease, which comprises administering a therapeutically effective amount of the protein according to the first aspect or the drug according to the seventh aspect The composition is administered to the subject. In one embodiment, the subject is a mammal, in another embodiment, the subject is a primate, and in another embodiment, the subject is a human.

 In one embodiment, the protein or pharmaceutical composition for preventing or treating rheumatoid arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, system Vasculitis, dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, lupus erythematosus, idiopathic thrombocytopenic purpura, glomerulonephritis, gout, rejection of organ transplants, Asthma or atherosclerosis.

 In one embodiment, the protein or pharmaceutical composition is for use in preventing or treating osteoarthritis, psoriatic joint inflammation, gouty arthritis, juvenile rheumatoid arthritis, septic arthritis.

 An eleventh aspect of the invention relates to a kit comprising the protein, gene, recombinant vector or host cell as described above.

 The experimental results of the present invention indicate that the protein can simultaneously block the B7/CD28 and TNFa/TNFR signaling pathways, and strongly inhibit the secretion of inflammatory cytokines in an in vitro experiment, and can effectively inhibit the development of joint inflammation in an animal model, and can achieve a composition ratio. The single functional unit of the protein can achieve a more therapeutic effect on the treatment of RA, that is, a partial superposition effect of the therapeutic effects of the two functional units. It is known to those skilled in the art that the combined use or fusion of several different mechanisms of action may result in different effects such as antagonism, irrelevance, accumulation or synergy, and the mutual use of two or more drugs in combination or fusion. The role can only be determined through specific experimental research and a lot of labor. Due to the complexity of the human body, especially the systemic and immune diseases such as RA, which are extremely complicated in pathogenesis and have not fully understood the pathogenesis, even if the drugs are used in combination or in combination, it is rare to achieve drugs with different mechanisms of action. A total additive or synergistic effect of the therapeutic effect, many of which exhibit a combination of irrelevant or even antagonistic effects. The enhanced therapeutic effect of RA achieved by the protein of the present invention provides a superior drug candidate for the treatment of RA patients, especially patients with moderate to severe RA. At the same time, the protein of the present invention simultaneously acts on two different targets for treating RA, which reduces the probability of failure or poor effect of single target treatment, and has important economic and social benefits. Compared with the case of using a single-function protein (for example, CTLA4-FC), the present invention can reduce the production cost, reduce the volume and frequency of clinical administration, improve subject compliance, and have a huge prevention and treatment of immune diseases. Application prospects. DRAWINGS

Figure 1 is a schematic diagram showing the structure of a bifunctional fusion protein of the present invention, wherein A is CTLA4-Fc-TNFR, and B is CTLA4-FC-anti-TNFascFv, C is CTLA4-TNFR-Fc, and D is CTLA4-anti-TNFascFv-Fc.

 Figure 2: A purification scheme for the recombinant bifunctional fusion protein of the present invention.

 Figure 3: Electrophoretic detection of recombinant bifunctional fusion proteins of the present invention. Lane 1 is reduced CTLA4-FC-TNFR, lane 2 is non-reduced CTLA4-Fc-TNFR, lane 3 is reduced CTLA4-Fc-anti-TNFascFv, and lane 4 is non-reduced CTLA4-Fc-anti-TNFascFv, Lane 5 is reduced CTLA4-TNFR-FC, lane 6 is non-reduced CTLA4-TNFR-Fc, lane 7 is reduced CTLA4-anti-TNFascFv-Fc, and lane 8 is non-reduced CTLA4-anti-TNFascFv-Fc.

 Figure 4: Binding of a recombinant bifunctional fusion protein to human TNFa in the present invention. A-C is the binding of CTLA4-FC-TNFR to human TNFa, the binding of CTLA4-Fc-anti-TNFa scFv to human TNFa, and the binding of four recombinant bifunctional fusion proteins of the invention to human TNFa, respectively.

 Figure 5: Kinetic constants for binding of recombinant bifunctional fusion proteins to mouse TNFa (A) and human TNFa (B) molecules in the present invention.

 Figure 6: Binding of the recombinant bifunctional fusion protein to the human CD80 (A) and human CD86 (B) molecules of the present invention. Figure Ί is the toxicity of recombinant CTLA4-Fc-TNFR, CTLA4-Fc-anti-TNFascFv bifunctional fusion protein and TNFa to L929 cells in the present invention.

 Figure 8 is a graph showing the results of inhibition of IL-2 expression by recombinant CTLA4-FC-TNFR in the MLR assay of the present invention. Fig. 9 shows the results of changes in the joint inflammation index of mice induced by CIA after CTLA4-FC-TNFR treatment in the present invention, and A and B respectively show the effect of inflammatory remission and body weight maintenance. detailed description

It is an object of the present invention to provide a bifunctional fusion protein, or abbreviated protein, which is capable of simultaneously blocking both B7/CD28 and TNFa/TNFR signaling pathways. Functional fragments that block the B7/CD28 signaling pathway are operably linked to functional segments that block the TNFa/TNFR signaling pathway, retaining their respective spatial structures and exerting their respective physiological activities. The functional fragment blocking the B7/CD28 signaling pathway and the functional fragment blocking the TNFa/TNFR signaling pathway may be directly fused together without affecting their respective functions, or may be between or at the end of the two functional fragments. Addition of other sequences, such as a linker peptide, or facilitating the two functional fragments to exert their respective activities, or to facilitate other biological effects, such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, Or an Fc fragment that facilitates the pharmacokinetic properties of the fusion protein, or other sequences that facilitate production and purification, such as antibodies. In one embodiment, the functional fragment that blocks the B7/CD28 signaling pathway is a CTLA4 molecule or a functional fragment thereof, and the term "CTLA4 molecule or a functional fragment thereof" as used herein refers to a cytotoxic T lymphocyte-associated antigen 4 ( Cytotoxic T lymphocyte-associated antigen 4, CTLA4 ) molecule and its functional fragment, the term "functional fragment" means The CTLA4 molecule functions as part of its function as a physiological function. In one embodiment, the functional fragment that blocks the TNFa/T FR signaling pathway is a functional fragment that neutralizes TNFa activity, and in the present invention, the term "functional fragment that neutralizes TNFa activity" means binding to TF a Any protein molecule that inactivates or partially inactivates the physiological function of TNFa. In a preferred embodiment, the functional fragment that neutralizes TNFa activity is T FR2 or a functional fragment thereof, and in the present invention, the term "TNFR2 or a functional fragment thereof" refers to TF receptor 2 or a functional fragment thereof, terminology "Functional fragment" refers to a portion of a TNFR2 molecule that functions as a physiological function. In an additional alternative embodiment, the functional fragment that neutralizes TNFa activity is an anti-TNFa antibody or a functional fragment thereof, and the term "functional fragment" refers to a portion of an anti-TNF a antibody that functions as a physiological function, such as a single structure. Domain antibody, single chain antibody, single chain variable fragment (scFv), Fab fragment or F(ab')2 fragment.

 Another object of the present invention is to provide a gene encoding the bifunctional fusion protein, a vector containing the gene, a host cell containing the vector, and a pharmaceutical composition containing the bifunctional fusion protein.

 It is still another object of the present invention to provide a method of preparing the bifunctional fusion protein.

 A further object of the invention is to provide the use of said bifunctional fusion protein.

 The bifunctional fusion protein provided by the invention has four combinations, one of which is the extracellular region of the CTLA4 molecule extracellular region-Fc fragment-linker peptide-T FR2 molecule, and the second is the CTLA4 molecule extracellular region-ligand peptide. - extracellular domain of TNFR2 molecule - Fc fragment, third is CTLA4 molecule extracellular domain - Fc fragment - linker peptide - anti-TNFa single chain antibody, and fourth is CTLA4 molecule extracellular domain - linker peptide - anti-TNFa Single-chain antibody-Fc fragment. It will be understood by those skilled in the art that although the qualifier used in the definition of the bifunctional fusion protein is "comprising", it does not mean that any other function unrelated to its function can be added to the bifunctional fusion protein sequence. sequence. In the preparation of a fusion protein of complex composition, in order to ensure the spatial structure, biological activity of the individual components of the fusion protein, and in order to properly fuse the various components together, or to enhance the hydrolysis resistance of the fusion protein, One skilled in the art, when preparing the fusion protein, will add one or more additional amino acid residues between the individual components or both ends of the fusion protein as needed, thus, if a closed expression is used to define The bifunctional fusion protein will not truly cover these situations.

In one embodiment, the functional fragment that blocks the B7/CD28 signaling pathway is the extracellular region of the CTLA4 molecule. The term "CTLA4 extracellular region" as used in the present invention refers to the extracellular region of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). According to a preferred embodiment, the amino acid sequence of the extracellular region of the CTLA4 molecule is set forth in SEQ ID NO: 1. Of course, on the basis of ensuring the physiological activity of the extracellular region of the CTLA4 molecule, the amino acid sequence thereof can correspondingly reduce or increase one or more amino acid residues along the amino acid sequence of positions 37-161 of the human CTLA4 protein. One or more amino acid residues in the amino acid sequence may also be substituted with conservative amino acids. Substitution of conservative amino acids is well known in the art. According to a preferred embodiment, the amino acid sequence of the Fc fragment is set forth in SEQ ID NO: 2. Of course, the amino acid sequence of the Fc fragment is also not unique, and it may be selected from the group consisting of Fc fragments of IgG, IgA, IgD, IgE, IgM, and combinations and hybrids thereof. One or more amino acid residues in the amino acid sequence may also be substituted with conservative amino acids.

 In certain embodiments, the term "functional fragment that blocks the T Fa / TNFR signaling pathway" as used herein refers to a TNF receptor, in one embodiment, the term "blocks a functional fragment of the TNFa/TNFR signaling pathway" Refers to TNF receptor 2 (TNFR2), in one embodiment, the term "functional fragment that blocks the TNFa/TNFR signaling pathway" refers to the extracellular region of TNFR2. According to a preferred embodiment, the amino acid sequence of the extracellular region of the T FR2 molecule is set forth in SEQ ID NO: 4. Of course, on the basis of ensuring the physiological activity of the extracellular region of the TNFR2 molecule, the amino acid sequence thereof can be correspondingly reduced or increased by one or more amino acid residues along the amino acid sequence 25-257 of the human T FR2 protein. One or more amino acid residues in the amino acid sequence may also be substituted with conservative amino acids.

 In certain embodiments, the term "functional fragment that blocks the TNFa/TNFR signaling pathway" as used herein refers to an immunoglobulin or a modification, functional equivalent, functional fragment or variant thereof that targets TNFa. In certain embodiments, the functional fragment that blocks the TNFa/TNFR signaling pathway is an IgG antibody (anti-TNFa monoclonal antibody) that targets TNFa. In certain embodiments, the IgG is a chimeric, humanized or fully human IgG. In certain embodiments, the modifications may be chemical modifications, such as acylation, guanidation, PEGylation products, so long as these modifications retain the ability to target TNFa. In certain embodiments, the functional equivalent refers to other polypeptide fragments that are capable of effecting the ability of the immunoglobulin to bind to TNFa. In certain embodiments, the functional fragment refers to a protein fragment that retains the ability to target TNFa, such as a single domain antibody, a single chain antibody, a single chain variable fragment (scFv), a Fab fragment, or F (ab) ') 2 fragment. In certain embodiments, the variant refers to a polypeptide derived from a parent protein by one or more alterations at one or more (several) positions, i.e., substitutions, insertions, and/or deletions.

 According to a preferred embodiment, the amino acid sequence of the anti-TNFa single-chain antibody is as shown in SEQ ID NO: 5. Of course, the amino acid sequence of the anti-TNFa single-chain antibody is also not unique, and it may be the amino acid sequence of any known single-chain antibody against TNFa. One or more amino acid residues in the amino acid sequence may also be substituted for a conservative amino acid.

In certain embodiments, the linker peptide is 1-50 amino acids in length, such as 5-45 amino acids, 10-35 amino acids, 15-20 amino acids, and in certain embodiments, the linker peptide The amino acid sequence is 1-25 amino acids in length. In one embodiment, the amino acid sequence of the linker peptide is 15 amino acids in length. In one embodiment, the amino acid sequence of the linker peptide is set forth in SEQ ID NO: 3. . The linker peptide used in the present invention is not special Restriction, as long as it functions as a spacer fusion protein, allows each component to correctly form its respective spatial structure, exert its biological activity, retain its cellular expression level and thermal stability.

 According to a preferred embodiment, the amino acid sequence of the bifunctional fusion protein of the invention is as shown in SEQ ID NOs: 6, 7, 8 and 9, or the sequences as shown in SEQ ID NOs: 6, 7, 8 and 9 are replaced. , an amino acid sequence having the same function formed by deleting or adding one or more amino acid residues, or an amino acid sequence having at least 70% identity and equivalent functions to the sequences of SEQ ID NO: 6, 7, 8 and 9 in one In an embodiment, the amino acid sequence of the bifunctional fusion protein of the invention has at least 80%, 85%, 90%, 95%, 96%, 97% of the sequence as set forth in SEQ ID NOs: 6, 7, 8 and 9. Amino acid sequences of 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7% or 99.8% identity and equivalent function.

 The term "identity" as used in the present invention has the meaning commonly known in the art, and those skilled in the art are also familiar with the rules and criteria for determining the identity between different sequences. Sequences defined by the present invention with varying degrees of identity must also have the extracellular domain of the CTLA4 molecule or neutralize IL-17 activity. Methods and means for screening variant sequences using the above activities are well known to those skilled in the art. Such variant sequences can be readily obtained by those skilled in the art under the teachings of the present disclosure.

 In another aspect, the invention provides a coding gene comprising a nucleotide sequence encoding a bifunctional fusion protein of the invention.

 It will be apparent to those skilled in the art that although the qualifier used in the definition of the coding gene is "included", it does not mean that any other sequence that is not related to its function can be arbitrarily added to both ends of the coding gene. It is known to those skilled in the art that in order to meet the requirements of the recombinant operation, it is necessary to add an appropriate restriction endonuclease cleavage site to both ends of the coding gene, or to additionally increase a promoter codon, a stop codon, etc., therefore, Defining the coding gene with a closed expression will not truly cover these situations.

 It is well known to those skilled in the art that one or more codons in the coding gene sequence can be replaced equidistantly, such as one or several codons, such as 1, 2, 3, without altering the encoded amino acid. , 4, 5, 6, 8, 9, 10, 15, 20, 30, 40, 50 codons. Codon usage tables are well known in the art.

 According to a preferred embodiment, the nucleotide sequence encoding the CTLA4-FC-TNFR of the present invention and the signal peptide sequence required for secretion expression is shown in SEQ ID NO: 10.

 According to a preferred embodiment, the nucleotide sequence encoding the CTLA4-T FR-FC of the present invention and the signal peptide sequence required for secretion expression is as shown in SEQ ID NO: 11.

According to a preferred embodiment, the nucleotide sequence encoding the CTLA4-FC-anti-F FascFv of the present invention and the signal peptide sequence required for secretion expression is set forth in SEQ ID NO: 12. According to a preferred embodiment, the nucleotide sequence encoding the CTLA4-anti-TNFascFv-Fc of the invention and the signal peptide sequence required for secretion expression is set forth in SEQ ID NO: 13. It is well known to those skilled in the art that one or more codons in the nucleotide sequence can be replaced isoformly without altering the encoded amino acid. Codon usage tables are well known in the art.

 In still another aspect, the invention provides a recombinant vector comprising a gene encoding a bifunctional fusion protein of the invention operably linked thereto. The recombinant vector is a recombinant expression vector, which may be a prokaryotic expression vector or a eukaryotic expression vector, but is preferably a eukaryotic expression vector, more preferably a recombinant expression vector for mammalian eukaryotic expression.

 The term "operably linked" as used in the present invention refers to a manner in which the coding gene is placed at a suitable position in the vector such that the coding gene is correctly and smoothly replicated, transcribed or expressed.

 In still another aspect, the invention provides a host cell comprising a vector comprising a gene encoding a bifunctional fusion protein of the invention. The host cell is a prokaryotic host cell or a eukaryotic host cell, but is preferably a eukaryotic host cell, more preferably a mammalian host cell. In one embodiment, the host cell comprises CHO cells, HEK293 cells, NSO cells, and SP 2/0 cells.

 In still another aspect, the present invention provides a method for producing the bifunctional fusion protein of the present invention, wherein the method comprises: (1) cloning the coding gene of the above aspect into a eukaryotic expression vector and transfecting into a host cell Expression; and (2) purification of the bifunctional fusion protein. Preferably, the purity of the purified bifunctional fusion protein is greater than 50%, more preferably greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

 In one embodiment, the eukaryotic expression vector is X0GC.

 In one embodiment, the host cell is HEK293-T and CHO.

 In still another aspect, the invention provides a pharmaceutical composition comprising a bifunctional fusion protein of the invention. The bifunctional fusion protein may be the sole active ingredient of the pharmaceutical composition or may be one of the active ingredients of the pharmaceutical composition, and the other active ingredient is another therapeutic agent that can be used in combination with the bifunctional fusion protein.

 In one embodiment, the pharmaceutical compositions of the present invention comprise a single administration dosage form, a topically administered dosage form, and a systemic administration dosage form.

 In still another aspect, the present invention provides a therapeutically effective amount of the bifunctional fusion protein or a pharmaceutical composition comprising the bifunctional fusion protein for preparing a medicament for preventing or treating an immune disease, a rejection, and a cardiovascular disease Use in.

 In one embodiment, the immune disease is selected from an autoimmune disease or an organ transplant disease.

In one embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriasis, type I diabetes, Multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, systemic vasculitis, dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, lupus erythematosus, special hair Thrombocytopenic purpura, glomerulonephritis, gout, rejection of organ transplants, asthma or atherosclerosis.

 In still another aspect, the present invention provides the bifunctional fusion protein or a pharmaceutical composition comprising the bifunctional fusion protein for use in preventing or treating an immune disease, a rejection reaction, and a cardiovascular disease.

 In one embodiment, the immune disease is selected from an autoimmune disease or an organ transplant disease.

 In one embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, systemic vasculitis, Dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, lupus erythematosus, idiopathic thrombocytopenic purpura, glomerulonephritis, gout, rejection of organ transplants, asthma or atherosclerosis hardening.

 In still another aspect, the present invention provides a method for preventing or treating an immune disease, a rejection, and a cardiovascular or cerebrovascular disease, comprising administering a therapeutically effective amount of the above aspect to a patient suffering from the disease or a population prone to the disease. The step of the bifunctional fusion protein or a pharmaceutical composition comprising the bifunctional fusion protein.

 In one embodiment, the immune disease is selected from an autoimmune disease or an organ transplant disease.

 In one embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmune encephalomyelitis, Crohn's disease, systemic vasculitis, Dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, lupus erythematosus, idiopathic thrombocytopenic purpura, glomerulonephritis, gout, rejection of organ transplants, asthma or atherosclerosis hardening.

 The term "therapeutically effective amount" as used in the present invention means a dose which can exert a pharmacological action in a subject upon administration. The "therapeutically effective amount" can be easily determined by a person skilled in the art depending on the condition of the patient such as age, body weight, disease state and the like.

 It is known to those skilled in the art that although the above-mentioned contents of the present invention list the conditions in which the bifunctional fusion protein of the present invention can prevent, treat or ameliorate, the conditions in which the bifunctional fusion protein of the present invention can be treated are not limited. The specific conditions listed above, any condition which can achieve a prophylactic, therapeutic or ameliorating benefit by simultaneously blocking both B7/CD28 and T Fa/TNFR signaling pathways are included within the scope of the present invention.

The bifunctional fusion protein provided by the present invention is capable of binding to the ligands CD80 and CD86 of CTLA4 and the ligand TNFa of TFR. In vitro experiments effectively neutralized the toxicity of TNFa to L929 cells and inhibited cell proliferation and inflammatory factor expression in experiments with human mixed lymphocyte reaction (MLR). Studies in mice that have been treated with CIA-induced arthritis have shown that the bifunctional fusion proteins provided by the present invention significantly ameliorate the progression of the disease. The structural form of the bifunctional fusion protein of the present invention is shown to retain the same biological activity as the native protein, and also The bifunctional fusion protein of the present invention has potential application value in immunomodulation, particularly immunosuppression. According to the above technical solution of the present invention, the present invention has the following advantageous effects:

 1) The blocking molecules of the two signaling pathways of B7 and TNFa (the extracellular domain of human CTLA4 and the extracellular domain of human TNFR2 or anti-TNFascFv) are fused into one molecule for expression and production, and the extracellular region producing CTLA4 protein, respectively. Compared with the extracellular region of human T FR2 or anti-TNFascFv, the method of operation and production cost are greatly reduced;

 2) The extracellular domain of human CTLA4 protein and the extracellular domain of human T FR2 or anti-TNFascFv are linked by the Fc fragment of the antibody, and the target protein forms a dimeric form, which satisfies both the CTLA4 extracellular domain and the T FR2 extracellular domain. The requirement to form a dimeric form to enhance activity also increases the potency of anti-TNFascFv, and in vitro binding experiments also show that the ligation mode of the present invention binds to the extracellular domain of CTLA4 molecule and its ligands CD80 and CD86, for T The extracellular domain of FR2 and the binding of anti-TNFascFv to TNFa have no effect;

 3) The results of CIA mouse model experiments show that the target bifunctional protein can effectively reduce the inflammatory response in mice. The invention will be further clarified by the following non-limiting examples, which are known to those skilled in the art, and many modifications may be made thereto without departing from the spirit of the invention.

 The following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified. The various antibodies used in the following examples of the invention are all derived from standard antibodies of the commercial route.

 Example

 Example 1: Recombinant CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc bifunctional fusion protein expression vector construction

 1) Amino acid sequence of CTLA4-FC-T FR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc

 The combined forms of CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc four bifunctional fusion proteins are shown in A-D of Figure 1.

The extracellular region sequence of the human CTLA4 protein is shown in SEQ ID NO: 1, which is the amino acid sequence of positions 37-161 of the human CTLA4 protein (Genbank Accession No. M_005214.4.). The Fc portion of IgG immunoglobulin (SEQ ID NO: 2) is referred to US Pat. No. 5,634,481. The linker peptide is (G ₄ S) ₃ (SEQ ID NO: 3). The extracellular region sequence of the TNFR2 protein is shown in SEQ ID NO: 4 and is the amino acid sequence 23-257 of the human TNFR2 protein (Genbank Accession No. P_001057.1). The anti-human TNFascFv sequence is shown in SEQ ID NO: 5, which is referred to in US20110002927A1.

 The amino acid sequence of CTLA4-FC-TNFR is shown in SEQ ID NO: 6, and the order from the N-terminus to the C-terminus is human.

The Fc portion of the extracellular domain of CTLA4 protein, IgG immunoglobulin, linker peptide (G ₄ S) ₃ and the extracellular domain of the human TNFR2 protein. The nucleotide sequence encoding the oncostatin-M signal peptide is shown in SEQ ID NO: 10. The amino acid of CTLA4-T FR-Fc is shown in SEQ ID NO: 7, and the sequence from the N-terminus to the C-terminus is the extracellular domain of human CTLA4 protein, the linker peptide (G ₄ S ) ₃ , the extracellular domain of human TNFR2 protein, and IgG. The Fc portion of an immunoglobulin. The nucleotide sequence encoding the oncostatin-M signal peptide is shown in SEQ ID NO: 11.

The amino acid of CTLA4-FC-anti-F FascFv is represented by SEQ ID NO: 8, and the sequence from the N-terminus to the C-terminus is the extracellular region of human CTLA4 protein, the Fc portion of IgG immunoglobulin, the linker peptide (G ₄ S ) ₃ and Anti-human TNFascFv. The nucleotide sequence encoding the oncostatin-M signal peptide is shown in SEQ ID NO: 12.

The amino acid of CTLA4-anti-TNFascFv-Fc is shown in SEQ ID NO: 9, and the sequence from the N-terminus to the C-terminus is the extracellular domain of human CTLA4 protein, the linker peptide (G ₄ S ) ₃ , the anti-TNFascFv and the IgG of the IgG immunoglobulin. section. The nucleotide sequence encoding the oncostatin-M signal peptide is shown in SEQ ID NO: 13.

 The above-mentioned coding nucleotide sequences were synthesized by Nanjing Kingsray Biotechnology Co., Ltd. and cloned into pUC57 vector by TA.

 2) Construction of CTLA4-Fc-T FR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc expression vector and preparation of transfection plasmid

 The pUC57-CTLA4-Fc-TNFR plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) was used as a template to amplify the CTLA4-FC-TNFR coding sequence by conventional PCR. The upstream primer used had a Hind III restriction site, and the sequence was CACAAGCTTGCCACCATGGGGGTCCTGCTGACTCAGAGG. (SEQ ID NO: 14). The downstream arch I carries a coR I restriction site, and the sequence is CCGGAATTCTCAGTCGCCAGTGCTCCC (SEQ ID NO:

15).

 The CTLA4-T FR-FC coding sequence was amplified by conventional PCR using pUC57-CTLA4-T FR-Fc plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) as a template, and the upstream primer used was SEQ ID NO: 14. The downstream primer carries the EcoR I restriction site and the sequence is CCGGAATTCTCACTTTCCTGGAGACAGG (SEQ ID NO: 16).

 The pUC57-CTLA4-Fc-anti-TNFascFv plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) was used as a template to amplify the CTLA4-FC-anti-TNFascFv coding sequence by conventional PCR. The upstream primer used had a Hind m restriction site. The upstream primer is SEQ ID NO: 14. The downstream primer carries the EcoR I restriction site and the sequence is CCGGAATTCTCAGCTGCTGACAGTGACCAGT (SEQ ID NO: 17).

The CTLA4-anti-TNFascFv-Fc coding sequence was amplified by conventional PCR using pUC57-CTLA4-anti-TNFascFv-Fc plasmid (Nanjing Kingsray Biotechnology Co., Ltd.) as a template, and the upstream primer used was SEQ ID NO: 14. The downstream primer is SEQ ID NO: 16. The amplified TLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc coding sequences were subjected to electrophoresis on a 1% agarose gel to recover the corresponding fragment. The recovered gene fragment and the company's eukaryotic expression vector X0GC (Patent US20100120089) were digested with Hind III and £coR I to obtain recombinant plasmids X0GC-CTLA4-Fc-TNFR, X0GC-CTLA4-TNFR-Fc, X0GC-CTLA4-Fc-anti-TNFascFv and X0GC-CTLA4-anti-T FascFv-Fc were transformed into E. coli DH5a, respectively, to obtain recombinant DH5a/X0GC-CTLA4-Fc-T FR, DH5a/X0GC-CTLA4-TNFR-Fc DH5a/X0GC-CTLA4-Fc-anti-TNFascFv and DH5a/X0GC-CTLA4-anti-TNFascFv-Fc. Positive clones were screened by PCR and subjected to DNA sequencing to verify that the recombinant plasmid was constructed correctly.

 Positive DH5a/X0GC-CTLA4-Fc-TNFR, DH5a/X0GC-CTLA4-T FR-Fc,

DH5a/X0GC-CTLA4-Fc-anti-TNFascFv and DH5a/X0GC-CTLA4-anti-TNFascFv-Fc were inoculated separately into IL LB/Amp liquid medium (composition of 1% peptone (BD company), 0.5% yeast extract (BD company) ), 1% NaCl (National Pharmaceutical Group Chemical Reagent Co., Ltd.)), shake culture at 37 ° C, 180 rpm overnight. The second angel was extracted with the DP117 endotoxin-free plasmid from Tiangen Biochemical Technology Co., Ltd. for transfection of HEK293-T and CHO cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). Example 2: Recombinant expression of CTLA4-Fc-TNFR, CTLA4-TNFR-Fc CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc bifunctional fusion protein

 1) Preparation of cell factory for HEK293-T cells

Grown in good condition, more than 95% confluent HEK293-T 18 cells>^<107 th to ten layers was inoculated cell factories (NUNC, Inc.) containing 10% fetal bovine serum (commercially available from Gibco, Inc.) DMEM medium (purchased from Coming) was cultured, and the cell factory was repeatedly inverted and mixed, and then cultured in a 37 ° C, 5% C0 ₂ incubator for 48 hours. The cell was completely adhered to a density of 80% for transient transfection. .

 2) Transient transfection and expression of HEK293-T cells

The X0GC-CTLA4-Fc-T FR, X0GC-CTLA4-TNFR-Fc, X0GC-CTLA4-Fc-anti-TNFascFv and X0GC-CTLA4-anti-TNFascFv-Fc recombinant plasmids were filtered through a 0.22 μηη filter, and then weighed 1330 μ§ The filtrate was added to 66 ml of serum-free DMEM medium. An equal volume of serum-free DMEM medium was added to 2660 μ _§ transfection reagent ΡΕΙ (purchased from Sigma), and then mixed with the plasmid filtrate, and allowed to stand for 15 minutes. The mixture containing the plasmid and PEI was added to 1.3 liters of serum-free DMEM medium, mixed well, and slowly added to the cell factory. The cell factory was cultured in a 37 ° C, 5% CO ₂ incubator. After 4 hours, 266 ml of Cell Boost 5 (purchased from Thermo Fisher Co., Ltd.) was added, and the mixture was further mixed for 3-4 days, and then centrifuged at 7000 rpm for 20 minutes to collect the supernatant for purification of the target protein.

 3) Preparation of cell factory for CHO-DG44 cells

Grown in good condition, more than 95% confluent CHO-DG44 cells in a 25><10 ⁷ to ten layers was inoculated in cell factories (NUNC, Inc.) containing 10% fetal bovine serum (commercially available from Gibco, Inc.) DMEM/F12 medium (purchased from Coming) was cultured, and the cell factory was repeatedly inverted and mixed, and then placed in a 37 ° C, 5% C0 ₂ incubator for 48 hours. When the cells are completely adherent and the density reaches 80%, they can be used for transient transfection.

 4) Transient transfection and expression of CHO-DG44 cells

The X0GC-CTLA4-Fc-T FR, X0GC-CTLA4 -TNFR-Fc, X0GC-CTLA4-Fc-anti-TNFascFv and X0GC-CTLA4-anti-T FascFv-Fc recombinant plasmids were filtered through a 0.22 μηη filter, and then weighed 1330μ. § Filtrate, add 66 ml of serum-free opti-MEM (purchased from Gibco) medium. An equal volume of serum-free opti-MEM medium was added to 2660 μ§ transfection reagent ΡΕΙ (purchased from Sigma), and then mixed with the plasmid filtrate, and allowed to stand for 15 minutes. The mixture containing the plasmid and PEI was added to 1.3 liters of serum-free opti-MEM medium, mixed well and slowly added to the cell factory. The cell factory was cultured in a 37 ° C, 5% CO ₂ incubator. After 4 hours, replace the culture medium in the factory with alpha-MEM (purchased from Coming) and add 266 ml Cell Boost 5 (purchased from Thermo Fisher). After mixing, in a 37 ° C, 5% C0 ₂ incubator The culture was cultured for 9 days. Thereafter, the supernatant was collected by centrifugation at 7000 rpm for 20 minutes for purification of the target protein. Example 3: Purification of recombinant CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc bifunctional fusion proteins

 The purification process of recombinant bifunctional protein is shown in Figure 2.

 1) Pretreatment of cell expression fermentation broth

 The harvested cell culture supernatant was centrifuged at 7000 rpm for 20 min to remove the precipitate. The supernatant of the cell fermentation broth was filtered through a 0.45 μm filter, concentrated by ultrafiltration at 30 Κ membrane and replaced with 20 mM PB buffer to add 150 mM sodium chloride, pH 7.4. The precipitate was removed by filtration through a 0.45 μη filter before applying column chromatography. This step was carried out at 4 °C.

 2) rProtein A affinity chromatography purification

 AKTA explorer 100 protein purification system (GE Healthcare) and affinity chromatography column rProtein A

Sepharose Fast Flow (16 mm ID., 10 ml, GE Healthcare) was purified at 4 °C. Mobile phase

A, 20 mM PB buffer was added to 150 mM sodium chloride, pH 7.4 solution equilibrium column, after the baseline was stabilized, the pretreated cell broth supernatant was loaded at a flow rate of 5 ml / min, and after loading Rinse with mobile phase A and elute with different buffers. First, wash 5 column volumes with mobile phase B1; then rinse 5 column volumes with mobile phase B2; then elute 5 column volumes with mobile phase B3, collect the elution peak as the target protein peak;

B4 rinses 5 column volumes. The flow rate of the above elution steps was 5 ml/min. Mobile phase B 1 is added with 0.5 M arginine in mobile phase A; mobile phase B2 is 20 mM NaAc, pH 4.5; mobile phase B3 is 100 mM citric acid, pH 3.0 _; mobile phase

B4 is lOOmM citric acid, pH 2.2. The indicated elution peaks were collected and the pH was adjusted to 5.0 by dropwise addition of 1 M NaAc.

 3) Protein ion exchange chromatography purification

AKTA explorer 100 protein purification system (GE Healthcare) and strong anion exchange chromatography column Purification was carried out at 4 °C using HiTrap Q Sepharose FF (5 ml, GE Healthcare). The column was first equilibrated with mobile phase A, a 20 mM NaAc (pH 5.0) solution. After the baseline was stabilized, the eluate collected and adjusted in the previous step was loaded at a flow rate of 5 ml/min. The flow through peak contains the protein of interest, and the flow through peak is collected and replaced into PBS buffer. The purity of the target protein was determined by SDS-PAGE, as shown in Figure 3. Example 4: Binding of recombinant CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc bifunctional fusion proteins to human TNFa

Recombinant human TNFa (GIBCO, Cat. No. PHC3015) at a concentration of 2 g/ml was coated on a 96-well high-adsorbing enzyme plate (Coming, 2592) in a volume of ΙΟΟμΙ/well, and reacted at a pH of 9.6 carbonate. 4 ° C overnight under buffer conditions. The next day, it was washed 5 times with PBST (Sigma, article number: P-3563). 300 μl of PBST containing 1% BSA was added to each well and blocked at 25 ° C for 1 hour. Wash 5 times with PBST. CTLA4-Fc-T FR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv, CTLA4-anti-TNFascFv-Fc, and positive control protein Etanercept (TNFR-Fc) (Wyeth) were formulated with PBST containing 1% BSA. Different concentrations were added to the microtiter plate at a ratio of ΙΟΟμΙ, and reacted at 25 ° C for 1 hour. Wash the paint 5 times with PBST. Horseradish peroxidase-labeled anti-human IgG antibody (Abeam, Cat. No.: Ab7153) was diluted with 3000 volumes of PBST containing 1% BSA and added to the plate in a ratio of ΙΟΟμΙ to react at 25 °C. hour. Wash PBST 5 times. ΙΟΟμΙ colorimetric substrate TMB (BD OptEIA, Cat. No.: 555214) was added to each well, and color development was carried out for 10 minutes at room temperature, and 显μΙ 1M H ₂ S0 ₄ was added to terminate the color development. The absorbance at 450 nm was read on a microplate reader. As a result, as shown by AC in Fig. 4, the binding activities of CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc to human TNFa were comparable to the control protein Etanetcept (TNFR-Fc). . Example 5: Kinetic constants of binding of recombinant CTLA4-FC-TNFR bifunctional fusion protein to mouse TNFa and human TNFa

 The BIAcore 3000 instrument was used to detect the kinetic constant of binding of the recombinant CTLA4-Fc-TNFR bifunctional fusion protein to its ligand TNFa. The instrument utilizes optical surface plasmon resonance techniques to detect the binding and dissociation between the molecules bound to the biochip and the molecule to be tested. All measurements were carried out at room temperature 25 °C.

CTLA4-Fc-T FR and Etanercept were coupled to a CM chip (GE Healthcare, BR-1000-50) via an amino coupling kit (GE Healthcare, BR-1000-14) with a coupling level of 2000 In response units, the flow rate was set to 10 μΙ7ηήη. The binding information of CTLA4-Fc-TNFR, Etanercept and its ligand molecule is obtained by multiple analysis cycles. For each analysis cycle, the flow rate was set to 20 μΙηη, and the injection time was 3 minutes (both mouse TNFa and human TNFa were diluted by 2 to obtain a series of different concentrations of antigen solution;), followed by dissociation for 5 minutes. . The regeneration conditions were 10 mM Gly-HCl solution, pH 2.0. Binding kinetic constants and dissociation kinetic constants BIAevaluation software calculation. The binding kinetic constant, dissociation kinetic constant and dissociation equilibrium constant of the recombinant CTLA4-Fc-T FR bifunctional fusion protein are shown in Figure 5. Example 6: Binding of recombinant CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv and CTLA4-anti-TNFascFv-Fc bifunctional fusion proteins to human CD80, CD86

Recombinant human CD80 (Sino Biological, Cat. No. 10698-H08H) and CD86 (Sino Biological, Cat. No. 10699-H08H) at a concentration of lg/ml were coated on a 96-well high-adsorbed ELISA plate according to the amount of ΙΟΟμΙ/well. The reaction was carried out overnight at 4 ° C under a carbonate buffer of ρ Η = 9.6. The next day, it was washed 5 times with PBST (Sigma, Cat. No. P-3563). 300 μl of PBST containing 1% BSA was added to each well and blocked at 25 ° C for 1 hour. Wash 5 times with PBST. CTLA4-Fc-T FR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-T FascFv, CTLA4-anti-TNFascFv-Fc, and positive control sample Abatacept (CTLA4-Fc) (Squibb) were configured with PBST containing 1% BSA Different concentrations were added to the microtiter plate in a ratio of ΙΟΟμΙ, and reacted at 25 ° C for 1 hour. Wash PBST 5 times. Horseradish peroxidase-labeled anti-human IgG antibody (Abeam, Cat. No.: Ab7153) was diluted with 3000 volumes of PBST containing 1% BSA and added to the plate in a ratio of ΙΟΟμΙ, and reacted at 25 °C. hour. Wash PBST 5 times. ΙΟΟμΙ Colorimetric substrate TMB (BD OptEIA, Cat. No. 555214) was added to each well, and color development was carried out for 10 minutes at room temperature, and 显μΙ 1M H ₂ S0 ₄ was added to terminate the color development. The absorbance at 450 nm was read on a microplate reader. As a result, as shown in A and B of Fig. 6, CTLA4-Fc-TNFR, CTLA4-TNFR-Fc, CTLA4-Fc-anti-TNFascFv, and CTLA4-anti-TNFascFv-Fc were all able to bind to human CD80 and CD86 molecules. Example 7: Neutralization of recombinant CTLA4-FC-TNFR and CTLA4-Fc-anti-TNFascFv bifunctional fusion protein

Toxicity of TNFa to L929 cells

The test proteins were diluted at various concentrations in RPMI-1640 medium (Gibco, Cat. No.: 22400) containing 2 g/ml actinomycin D, 4 ng/ml human TNFa and 10% fetal bovine serum (Gibco, Cat. No. 10099). ) in standby. L929 cells (purchased from ATCC) were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and adjusted to a cell concentration of 1.5 χ 10 ⁵ /ml, and ΙΟΟμΙ^ was added to a flat-bottom 96-well plate at 37°. C, 5% C0 ₂ in the cell incubator, discard the supernatant after 24 hours, add fresh medium and test protein 50μ1, mix and incubate in 37 ° C, 5% C0 ₂ cell incubator for 24 hours. . MTS (Promega, G3581) was added in an amount of 20 μl/well and incubation was continued for 3 hours. The absorbance at 490 nm was read on a microplate reader. As a result, as shown in Fig. 7, CTLA4-Fc-TNFR and CTLA4-Fc-anti-TNFascFv effectively neutralized the killing effect of TNFa on L929 cells. Example 8: Recombinant CTLA4-FC-TNFR bifunctional fusion protein inhibits IL-2 expression

Human PBMCs were provided by two healthy volunteers. Whole blood was diluted with DPBS at a ratio of 1:1. In the centrifuge tube Add human lymphocyte separation solution at room temperature, gently add a mixture of blood and DPBS in a ratio of 1:1, centrifuge at 400g for 20 minutes, remove the second layer of milky white cells, add 1 to 5 DPBS, centrifuge at 10 rpm for 10 minutes. PBMCs. The DPBS was washed twice, resuspended in RPMI 1640 containing 10% FBS, and counted. One of the volunteer-derived PBMCs was used as a stimulator and treated with mitomycin C (5 (^g/ml) for 45 minutes at 37 ° C. Another volunteer-derived PBMCs served as responders. The responder PBMCs and mitomycin C-treated stimulator PBMCs were added to a round-bottom 96-well plate, each lx lO ⁵ cells/well, 50 μl/well. The cells were mixed and then ΙΟΟμΙ gradient-diluted CTL A4-Fc- was added. T FR, CTLA4-Fc, Humira (Abbott) or DPBS buffer, three replicates per sample.

 The MLR-reacted cells were incubated in a 37 ° C incubator for 3 days, and 50 μl of the cell supernatant was taken out. The IL-2 ELISA test kit (purchased from RayBiotech, article number ELH-IL-2-001) was used to detect IL-2. expression. Results As shown in Fig. 8, CTLA4-FC-T FR has an activity of inhibiting IL-2 expression, and the activity is superior to that of CTLA4-Fc or Humira alone at the same concentration. Example 9: Pharmacological effect of recombinant CTLA4-FC-TNFR bifunctional fusion protein in type II collagen-induced mouse arthritis (CIA) model

Eight-week-old male DBA1/J mice (purchased from Shanghai Slack Laboratory Animals Co., Ltd.) were randomly divided into two groups, one group of 7 was used as a normal mouse control, and the remaining mice were divided into another group to establish a CIA model. The CIA mouse model was established by two immunizations, primary immunization and booster immunization. The primary immunization was mixed with 70 μ _§ Type II bovine collagen (Chondrex, Cat. No. 20022) and Freund's complete adjuvant (Sigma-Aldrich, Cat. No. F5881) to form an emulsion, which was injected intradermally into the tail of the mouse. After three weeks, strengthen the immunization. The booster immunization was performed by mixing 70 μ _§ II bovine collagen with Freund's incomplete adjuvant (Sigma-Aldrich, Cat. No. F5506) to form an emulsion, which was injected intradermally into the tail of the mouse. After clinically induced arthritis symptoms such as swelling of the limbs of the mice were observed after booster immunization, CIA model mice were randomly divided into groups of 7 mice each receiving vehicle or drug. Dosing samples and doses were Abatacept (33 nmol/kg;), Etanercept (33 nmol/kg;), combined administration of Abatacept (33 nmol/kg) and Etanercept (33 nmol/kg) and CTLA4-Fc-TNFR (33 nmol/kg) o It was administered once every two days by intraperitoneal injection, and was administered on days 0, 2, 4, 6, and 8, respectively, for a total of 5 times. Weigh the body weight, observe the lesions of the limbs before and after the limbs, and score the arthritis index: 0 = no redness, 1 = erythema or patella joints with erythema, slight swelling, 2 = erythema from the ankle to the tibia Slight swelling, 3 = erythema from the ankle to the tibial joint, moderate swelling, 4 = ankle joint, paws including the phalanx joints have erythema, severe swelling or joint stiffness of the limbs. As a result, as shown in Fig. 9, the arthritis index of CIA-induced mice decreased significantly after CTLA4-FC-TNFR treatment, which was better than the therapeutic effects of Aatacept, Etanercept and co-administration at the same concentration, showing good inflammatory remission ( Figure A); and also shows a better weight maintenance effect (Figure B).

Claims

Rights request 1. A protein whose amino acid sequence is selected from the following sequences: a) The sequence shown in SEQ ID NO: 6, 7, 8 or 9; b) A sequence formed by replacing, deleting or adding one or more amino acid residues to the sequence shown in SEQ ID NO: 6, 7, 8 or 9 and having the activity of simultaneously blocking the B7/CD28 and TNFa/T FR signaling pathways. ; or c) A sequence that has at least 70% identity with the sequence shown in SEQ ID NO: 6, 7, 8 or 9 and has the activity of simultaneously blocking the B7/CD28 and TNFa/TNFR signaling pathways. 2. A gene encoding the protein according to claim 1. 3. The gene according to claim 2, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 10, 11, 12 or 13. 4. A recombinant vector to which the gene according to claim 3 is effectively connected. 5. A host cell containing the recombinant vector according to claim 4. 6. A method for preparing the protein of claim 1, said method comprising: 1) Clone the gene described in claim 2 into a eukaryotic expression vector and transfect it into a host cell for expression; and 2) purify the protein, Preferably, the eukaryotic expression vector is XOGC, and preferably, the host cells are HEK293-T and CHO. 7. A pharmaceutical composition containing the protein of claim 1. 8. Use of a protein according to claim 1 or a pharmaceutical composition according to claim 7 in the preparation of drugs for preventing or treating immune diseases, rejection reactions and cardiovascular and cerebrovascular diseases. 9. The use according to claim 8, characterized in that the protein is used to prepare medicines for preventing or treating the following diseases: rheumatoid arthritis, psoriasis, type I diabetes, multiple sclerosis, autoimmunity Encephalomyelitis, Crohn's disease, systemic vasculitis, dermatomyositis, mixed connective tissue disease, ankylosing spondylitis, psoriatic arthritis, Lupus erythematosus, idiopathic thrombocytopenic purpura, glomerulonephritis, gout, organ transplant rejection, asthma or atherosclerosis. 10. The use according to claim 8 or 9, characterized in that the protein is used to prepare drugs for preventing or treating the following diseases: osteoarthritis, psoriatic arthritis, gouty arthritis, juvenile arthritis Rheumatoid arthritis, septic arthritis.